Peptides (v.29, #8)

Identification of a novel storage glycine-rich peptide from guava (Psidium guajava) seeds with activity against Gram-negative bacteria by Patricia B. Pelegrini; André M. Murad; Luciano P. Silva; Rachel C.P. dos Santos; Fabio T. Costa; Paula D. Tagliari; Carlos Bloch Jr; Eliane F. Noronha; Robert N.G. Miller; Octavio L. Franco (1271-1279).
Bacterial pathogens cause an expressive negative impact worldwide on human health, with ever increasing treatment costs. A significant rise in resistance to commercial antibiotics has been observed in pathogenic bacteria responsible for urinary and gastro-intestinal infections. Towards the development of novel approaches to control such common infections, a number of defense peptides with antibacterial activities have been characterized. In this report, the peptide Pg-AMP1 was isolated from guava seeds (Psidium guajava) and purified using a Red-Sepharose Cl-6B affinity column followed by a reversed-phase HPLC (Vydac C18-TP). Pg-AMP1 showed no inhibitory activity against fungi, but resulted in a clear growth reduction in Klebsiella sp. and Proteus sp., which are the principal pathogens involved in urinary and gastro-intestinal hospital infections. SDS-PAGE and mass spectrometry (MALDI-ToF) characterized Pg-AMP1 a monomer with a molecular mass of 6029.34 Da and small quantities of a homodimer. Amino acid sequencing revealed clear identity to the plant glycine-rich protein family, with Pg-AMP1 the first such protein with activity towards Gram-negative bacteria. Furthermore, Pg-AMP1 showed a 3D structural homology to an enterotoxin from Escherichia coli, and other antibacterial proteins, revealing that it might act by formation of a dimer. Pg-AMP1 shows potential, in a near future, to contribute to development of novel antibiotics from natural sources.
Keywords: Psidium guajava; Toxin; Glycine-rich proteins; Antimicrobial peptides;

A bradykinin (BK)-related peptide was isolated from heat-denaturated plasma from an elasmobranch fish, the little skate, Leucoraja erinacea after incubation with porcine pancreatic kallikrein. The primary structure of the peptide (H-Gly-Ile-Thr-Ser-Trp-Leu-Pro-Phe-OH; skate BK) shows limited structural similarity to the mammalian B1 receptor agonist, des-Arg9-BK. The myotropic activities of synthetic skate BK, and the analog skate [Arg9]BK, were examined in isolated skate vascular and intestinal smooth muscle preparations. Skate BK produced a concentration-dependent constriction of the mesenteric artery (EC50  = 4.37 × 10−8  M; maximum response = 103.4 ± 10.23% of the response to 60 mM KCl) but the response to skate [Arg9]BK was appreciably weaker (response to 10−6  M = 73.0 ± 23.4% of the response to 60 mM KCl). Neither the first branchial gill arch nor the ventral aorta responded to either purified peptide. Skate BK also produced a concentration-dependent constriction of intestinal smooth muscle preparations (EC50  = 2.74 × 10−7  M; maximum response 31.0 ± 12.2% of the response to 10−5  M acetylcholine). Skate [Arg9]BK was without effect on the intestinal preparation. The data provide evidence for the existence of the kallikrein–kinin system in a phylogenetically ancient vertebrate group and the greater potency of skate BK compared with the analog skate [Arg9]BK suggests that the receptor mediating vascular responses resembles the mammalian B1 receptor more closely than the B2 receptor.
Keywords: Elasmobranch; Skate; Bradykinin; B1 receptor; Kallikrein–kinin system;

Purification and characterization of antimicrobial peptides from the Caribbean frog, Leptodactylus validus (Anura: Leptodactylidae) by Jay D. King; Jérôme Leprince; Hubert Vaudry; Laurent Coquet; Thierry Jouenne; J. Michael Conlon (1287-1292).
Peptidomic analysis of norepinephrine-stimulated skin secretions from the Caribbean frog Leptodactylus validus Garman, 1888 led to the identification of three peptides with previously undescribed sequences that were structurally similar to those of antimicrobial peptides isolated from other species of leptodactylid frogs. These paralogs have been termed ocellatin-V1 (GVVDILKGAGKDLLAHALSKLSEKV.NH2), ocellatin-V2 (GVLDILKGAGKDLLAHALSKISEKV.NH2), and ocellatin-V3 (GVLDILTGAGKDLLAHALSKLSEKV.NH2). The very low antimicrobial potency (MIC > 200 μM) against Escherichia coli and Staphylococcus aureus associated with the peptides is probably a consequence of their lack of amphipathicity and reduced cationicity compared with active members of the ocellatin family from related species.
Keywords: Amphipathic α-helix; Antimicrobial; Frog skin; Ocellatin;

Crotalphine, a novel potent analgesic peptide from the venom of the South American rattlesnake Crotalus durissus terrificus by Katsuhiro Konno; Gisele Picolo; Vanessa P. Gutierrez; Patrícia Brigatte; Vanessa O. Zambelli; Antonio C.M. Camargo; Yara Cury (1293-1304).
We have shown that the venom of the South American rattlesnake Crotalus durissus terrificus induces a long-lasting antinociceptive effect mediated by activation of κ- and δ-opioid receptors. Despite being mediated by opioid receptors, prolonged treatment with the crotalid venom does not cause the development of peripheral tolerance or abstinence symptoms upon withdrawal. In the present study, we have isolated and chemically characterized a novel and potent antinociceptive peptide responsible for the oral opioid activity of this crotalid venom. The amino acid sequence of this peptide, designated crotalphine, was determined by mass spectrometry and corroborated by solid-phase synthesis to be <EFSPENCQGESQPC, where <E is pyroglutamic acid and the two cysteine residues forming a disulfide bond. This 14-amino-acid residue sequence is identical to the γ-chain sequence of crotapotin, a non-toxic component of this snake venom. Crotalphine, when orally administered (0.008–25 μg/kg), induces antinociceptive effect in the prostaglandin E2- and carrageenin-induced mechanical hyperalgesia models in rats and in the hot-plate test in mice. Crotalphine was also effective when administered by intravenous (0.0032–0.04 μg/kg) or intraplantar (s.c., 0.00006–0.3 μg/paw) routes. In the mechanical hyperalgesia models, crotalphine shows a long-lasting (5 days) antinociceptive effect. d-Phe-Cys-Tyr-d-Trp-Orn-Thr-Pen-Thr amide (CTOP) and N,N-diallyl-Tyr-Aib-Aib-Phe-Leu (ICI 174,864), antagonists of μ- and δ-opioid receptors, respectively, did not alter the antinociceptive effect of the peptide, whereas nor-binaltorphimine, an antagonist of κ-opioid receptors, blocked this effect. These results indicate that crotalphine induces antinociception mediated by activation of κ-opioid receptors and may contribute to the antinociceptive effect of the crotalid venom.
Keywords: Crotalphine; Antinociception; Opioid receptor; Pain; Snake venom;

The synthesis and anticancer activity of selected diketopiperazines by E. van der Merwe; D. Huang; D. Peterson; G. Kilian; P.J. Milne; M. Van de Venter; C. Frost (1305-1311).
Six selected diketopiperazines, cyclo(Gly-Val), cyclo(Gly-D-Val), cyclo(Gly-Leu), cyclo(Gly-Ile), cyclo(Phe-Cys) and cyclo(Tyr-Cys), were synthesized via various synthetic routes. Their potential to inhibit cancer cell growth in HT-29, HeLa and MCF-7 cells was determined. Cyclo(Tyr-Cys) caused the greatest inhibition in cervical carcinoma cells with near equivalent activity against HT-29 and MCF-7 cells. The other cyclic dipeptides tested were effective in the inhibition of colon, cervical and breast carcinoma cells, respectively, but the percentage inhibition was lower than for cyclo(Tyr-Cys).
Keywords: Cyclic dipeptides; Cancer cells; MCF-7; HT-29; HeLa;

The angiotensin converting enzyme inhibitory tripeptides Ile-Pro-Pro and Val-Pro-Pro show increasing permeabilities with increasing physiological relevance of absorption models by Martin Foltz; Anja Cerstiaens; Ans van Meensel; Raf Mols; Pieter C. van der Pijl; Guus S.M.J.E. Duchateau; Patrick Augustijns (1312-1320).
Transepithelial transport of the ACE inhibitory peptides Ile-Pro-Pro and Val-Pro-Pro was studied in different models of absorption. Apparent permeability (P app) values for absorptive transport across Caco-2 monolayers were 1.0 ± 0.9 × 10−8 (Ile-Pro-Pro) and 0.5 ± 0.1 × 10−8  cm s−1 (Val-Pro-Pro). Ex vivo transport across jejunal segments in the Ussing chamber was 5-times (Ile-Pro-Pro) to 10-times (Val-Pro-Pro) higher with no significant differences (p  > 0.05) observed between both peptides. The peptidase inhibitor bestatin increased permeability for the absorptive direction for Ile-Pro-Pro by twofold. Neither a transepithelial pH gradient nor increased apical tripeptide concentration nor longitudinal localization of the intestinal segment influenced P app in the ex vivo experiments. Val-Pro-Pro transport across Peyer's patches, however, was 4-times higher (P app  = 21.0 ± 9.3 × 10−8  cm s−1) as compared to duodenum (P app  = 4.8 ± 1.4 × 10−8  cm s−1). In the in situ perfusion experiments P app values varied greatly among different animals ranging from 0.5 to 24.0 × 10−8  cm s−1 (Ile-Pro-Pro) and from 1.0 to 15.6 × 10−8  cm s−1 (Val-Pro-Pro). In summary, Caco-2 and ex vivo absorption models differ considerably regarding their peptide permeability. The in situ model seems to be less appropriate because of the observed large variability in peptide permeability. The results of this study demonstrate that the ACE inhibitory peptides Ile-Pro-Pro and Val-Pro-Pro are absorbed partially undegraded.
Keywords: Absorption models; ACE inhibitory peptides; apparent permeability; peptide transport;

The present study was designed to investigate the effects of rat intermedin/adrenomedullin2 (rIMD), an agonist for calcitonin-like calcitonin receptors (CRLR), on the isolated rat pulmonary arterial rings (PA). When PA were precontracted with 9,11-dideoxy-11α,9α-epoxymethanoprostaglandin F2α (U-46619), rIMD (10−11 to 10−6  M) induced concentration-dependent relaxation. The pulmonary vasorelaxant response (PVR) to rIMD in PA were completely inhibited by endothelium removal, NG-nitro-l-arginine-methyl-ester (l-NAME), l-N5-(1-iminoethyl)-ornithine hydrochloride (l-NIO) or 1H-[1,2,4] oxadiazolo[4,3-a]quinoxalin-1-one (ODQ). The PVR to rIMD were also significantly attenuated by a protein kinase inhibitor, Rp-8-bromo-β-phenyl-1,N2-ethenoguanosine 3′:5′-cyclic monophosphorothioate sodium salt hydrate (Rp-8-Br-PETcGMPs), cholera toxin and abolished by tetraethylammonium chloride (TEA), iberiotoxin and precontraction with KCl. The relaxant effect was not affected by 9-(tetrahydro-2-furanyl)-9H-purin-6-amine (SQ22536), (9S,10S,12R)-2,3,9,10,11,12-hexahydro-10-hydroxy-9-methyl-1-oxo-9,12-epoxy 1H diindolo [1,2,3fg:3′,2′,1′kl] pyrrolo [3,4-i] [1,6] benzodiazocine-10-carboxylic acid hexyl ester (KT5720), meclofenamate, glybenclamide or apamin. In parallel with SQ22536 and KT5720 results rolipram pretreatment did not alter the rIMD-induced PVR. The PVR to rIMD was potentialized either in the presence of zaprinast or sildenafil. Since the PVR to rIMD was also significantly reduced by rCGRP8–37 and hADM22–52 and rIMD17–47, the present data suggest that rIMD produces PVR by acting in an indiscriminant manner on functional, and possibly different, endothelial CRLR. In conclusion, rIMD stimulates endothelial CRLR are coupled to release of nitric oxide, activation of guanylate cyclases, and promotion of hyperpolarization through large conductance calcium-activated K+ channels in rat main PA.
Keywords: Intermedin/adrenomedullin2; Isolated pulmonary artery; Nitric oxide; BKCa Channels;

The role of ETA and ETB receptor antagonists in acute and allergic inflammation in mice by Cândida A.L. Kassuya; Alexandre P. Rogerio; João B. Calixto (1329-1337).
In this study, we investigated the effects of the selective ETA (BQ-123) and ETB (BQ-788) receptor antagonists for endothelin-1 (ET-1) against several flogistic agent-induced paw edema formation and ovalbumin-induced allergic lung inflammation in mice. The intraplantar injection of BQ-123, but not BQ-788, significantly inhibited carrageenan-, PAF-, ET-1- and bradykinin-induced paw edema formation. The obtained inhibitions (1 h after the inflammatory stimulus) were 79 ± 5%, 55 ± 4%, 55 ± 6% and 74 ± 4%, respectively. In carrageenan-induced paw edema, the mean ID50 value for BQ-123 was 0.77 (0.27–2.23) nmol/paw. The neutrophil influx induced by carrageenan or PAF was reduced by BQ-123, with inhibitions of 55 ± 2% and 72 ± 4%, respectively. BQ-123 also inhibited the indirect macrophage influx induced by carrageenan (55 ± 6%). However, BQ-788 failed to block the cell influx caused by either of these flogistic agents. When assessed in the bronchoalveolar lavage fluid in a murine model of asthma, both BQ-123 and BQ-788 significantly inhibited ovalbumin-induced eosinophil recruitment (78 ± 6% and 71 ± 8%), respectively. Neither neutrophil nor mononuclear cell counts were significantly affected by these drugs. Our findings indicate that ETA, but not ETB, selective ET-1 antagonists are capable of preventing the acute inflammatory responses induced by carrageenan, PAF, BK and ET-1. However, both ETA and ETB receptor antagonists were found to be effective in inhibiting the allergic response in a murine model of asthma.
Keywords: Endothelin-1; Endothelin receptor antagonists; Inflammation; Asthma;

Gender differences in vascular reactivity to endothelin-1 (1-31) in mesenteric arteries from diabetic mice by Takayuki Matsumoto; Mika Kakami; Tsuneo Kobayashi; Katsuo Kamata (1338-1346).
Endothelin-1 (1-31) [ET-1 (1-31)], a novel member of the ET family, comprises 31 amino acids and is derived from the selective hydrolysis of big ET-1 by chymase. Although ET-1 (1-31) reportedly exerts biological effects by direct or indirect [via its conversion to ET-1 (1-21)] mechanisms, it is unclear whether in diabetes the vascular effects of ET-1 (1-31) display gender differences. We investigated this question by exposing mesenteric artery rings to ET-1 (1-31), using arteries from mice in the early or chronic phase of diabetes. In the early stage of diabetes, the ET-1 (1-31)-induced contraction was similar between age- and sex-matched control and streptozotocin (STZ)-induced diabetic mice. In the chronic stage of diabetes, the ET-1 (1-31)-induced contraction was enhanced in diabetic female mice, but not in diabetic male mice (vs. both age-matched control and early-stage diabetic mice). This enhancement was largely prevented by Y27632 (Rho kinase inhibitor), PD98059 [inhibitor of extracellular signal related kinases 1 and 2 (ERK1/2)], or SP600125 [C-jun terminal kinase (JNK) inhibitor]. These data indicate that the ET-1 (1-31)-induced vasoconstriction in the mesenteric artery may be specifically enhanced in established diabetic female mice, and that this enhancement may be due to alterations in the activities of Rho/Rho kinase or mitogen-activated protein kinase.
Keywords: Contraction; Diabetes; Gender difference; Endothelin; Mesenteric artery; Mice;

Long-term administration of maxadilan improves glucose tolerance and insulin sensitivity in mice by Rongjie Yu; Tianhong Yi; Shanshan Xie; An Hong (1347-1353).
Maxadilan and its truncated variant, M65, are agonist and antagonist specific, respectively, for the PAC1 receptor. PAC1 is the specific receptor for the pituitary adenylate cyclase-activating peptide (PACAP), which is not shared by vasoactive intestinal peptide (VIP). PACAP is a ubiquitous peptide of the glucagon superfamily that is involved in glucose homeostasis and regulation of insulin secretion. This study employed the recombinant maxadilan and M65 to evaluate the PAC1 receptor-mediated effects on energy metabolism using NIH mice. First, the acute effect of maxadilan-induced hyperglycemia was blocked by M65. In long-term studies, NIH mice were given daily intraperitoneal injections with maxadilan, M65, or vehicle for 21 days. Maxadilan suppressed feeding and enhanced water intake significantly for the first several days. After that period, maxadilan treatment continued to promote food and water intake. Long-term administration of maxadilan led to an increase in body weight (P  < 0.01), decrease in body fat (P  < 0.01), down-regulation of basal plasma glucose (P  < 0.01), upregulation of basal plasma insulin (P  < 0.01) and improved glucose tolerance (P  < 0.01) and insulin sensitivity (P  < 0.01). An elevation in plasma LDL (P  < 0.01) was also observed in the maxadilan group. However, M65 displayed no significant adverse effects on the aforementioned parameters except basal plasma glucose (P  < 0.05). The significant changes induced by maxadilan indicate that the PAC1 receptor plays multiple key roles in carbohydrate metabolism, lipid metabolism and energy homeostasis in mice.
Keywords: Maxadilan; PACAP; PAC1; Glucose; Insulin;

Metabolic effects of chronic obestatin infusion in rats by Suraj Unniappan; Madeleine Speck; Timothy J. Kieffer (1354-1361).
Obestatin is purported to be a peptide hormone encoded in preproghrelin. We studied the metabolic effects of continuous infusion of obestatin via subcutaneously implanted osmotic mini-pumps. Administration of up to 500 nmol/kg body weight/day obestatin did not change 24 h cumulative food intake or body weight in rats. Similarly, no effects were observed when obestatin was infused at 1000 nmol/kg body weight/day for seven days. This dose of obestatin infused during a 24 h fast did not alter weight loss, suggesting that obestatin has no effect on energy expenditure, and this dose did not alter glucose or insulin responses during an IPGTT. Obestatin was originally proposed to interact with GPR39 and subsequently the receptor for GLP-1. While both receptors are expressed in pancreatic islets, incubation with obestatin did not alter insulin release from islets in vitro. Moreover, obestatin did not bind to INS-1 β-cells or HEK cells overexpressing GLP-1 receptors or displace GLP-1 binding to these cells. Our findings do not support the concept that obestatin is a hormone with metabolic actions.
Keywords: Food intake; Water intake; Energy Balance; Body weight; Glucose homeostasis; Osmotic mini-pumps; GLP-1;

Effects of chronic food restriction and treatments with leptin or ghrelin on different reproductive parameters of male rats by Alexander V. Sirotkin; Maria Chrenková; Soňa Nitrayová; Peter Patraš; Krzysztof Darlak; Francisco Valenzuela; Leonor Pinilla; Manuel Tena-Sempere (1362-1368).
The existence of a close relationship between energy status and reproductive function is well-documented, especially in females, but its underlying mechanisms remain to be fully unfolded. This study aimed to examine the effects of restriction of daily calorie intake, as well as chronic treatments with the metabolic hormones leptin and ghrelin, on the secretion of different reproductive hormones, namely pituitary gonadotropins and prolactin, as well as testosterone, in male rats. Restriction (50%) in daily food intake for 20 days significantly reduced body weight as well as plasma PRL and T levels, without affecting basal LH and FSH concentrations and testicular weight. Chronic administration of leptin to rats fed ad libitum increased plasma PRL levels and decreased circulating T, while it did not alter other hormonal parameters under analysis. In contrast, in rats subjected to 50% calorie restriction, leptin administration increased plasma T levels and reduced testis weight. Conversely, ghrelin failed to induce major hormonal changes but tended to increase testicular weight in fed animals, while repeated ghrelin injections in food-restricted males dramatically decreased plasma LH and T concentrations and reduced testis weight. In sum, we document herein the isolated and combined effects of metabolic stress (50% food restriction) and leptin or ghrelin treatments on several reproductive hormones in adult male rats. Overall, our results further stress the impact and complex way of action of different metabolic cues, such as energy status and key hormones, in reproductive function also in the male.
Keywords: Feeding; Growth; Leptin; Ghrelin; Testis; Gonadotropins; Prolactin; Testosterone;

Role of ghrelin axis in colorectal cancer: A novel association by Talat Waseem; Javaid-ur-Rehman; Farooq Ahmad; Muhammad Azam; Mansoor Ahmad Qureshi (1369-1376).
Recently discovered orexigenic peptide, ghrelin, which is primarily produced by gastrointestinal tract, has been implicated in the malignant cell proliferation and invasion, presumably through an autocrine/paracrine mechanism. This study was aimed to identify the role of endogenously produced ghrelin in colorectal cancer progression. Malignant intestinal epithelial cells differentially over-express ghrelin receptors and produce more ghrelin as compared to normal human colonocytes, leading to their enhanced proliferative and invasive behavior. Though, systemically available endocrine ghrelin levels in patients with colorectal cancer do not exhibit significant correlation with any tumor stage or grade, however, locally produced autocrine tissue ghrelin strongly correlates both with advancing colorectal malignancy in a stage-dependent manner and BMI of the colorectal patients. We conclude that ghrelin might play an important role in promoting colorectal malignancy.
Keywords: Ghrelin; Colorectal cancer; Autocrine/paracrine mechanism; Cell proliferation;

Glucagon-like peptide 1 (7–36) amide (GLP-1) and exendin-4 stimulate serotonin release in rat hypothalamus by Luigi Brunetti; Giustino Orlando; Lucia Recinella; Sheila Leone; Claudio Ferrante; Annalisa Chiavaroli; Francesco Lazzarin; Michele Vacca (1377-1381).
Glucagon-like peptide 1 (7–36) amide (GLP-1) and exendin-4 are gastrointestinal hormones as well as neuropeptides involved in glucose homeostasis and feeding regulation, both peripherally and at the central nervous system (CNS), acting through the same GLP-1 receptor. Aminergic neurotransmitters play a role in the modulation of feeding in the hypothalamus and we have previously found that peripheral hormones and neuropeptides, which are known to modulate feeding in the central nervous system, are able to modify catecholamine and serotonin release in the hypothalamus. In the present paper we have evaluated the effects of GLP-1 and exendin-4 on dopamine, norepinephrine, and serotonin release from rat hypothalamic synaptosomes, in vitro. We found that glucagon-like peptide 1 (7–36) amide and exendin-4 did not modify either basal or depolarization-induced dopamine and norepinephrine release; on the other hand glucagon-like peptide 1 (7–36) amide and exendin-4 stimulated serotonin release, in a dose dependent manner. We can conclude that the central anorectic effects of GLP-1 agonists could be partially mediated by increased serotonin release in the hypothalamus, leaving the catecholamine release unaffected.
Keywords: Glucagon-like peptide 1 (GLP-1); Exendin-4; Hypothalamus; Dopamine; Norepinephrine; Serotonin;

Visfatin expression is elevated in normal human pregnancy by S.A. Morgan; Jonathon B. Bringolf; E.R. Seidel (1382-1389).
Visfatin is a novel-secreted 52 kDa adipokine that appears to mimic the action of insulin, inducing glucose transport into mammalian cells. We examined visfatin expression in a cohort of pregnant women to determine if pregnancy influenced visfatin gene expression, circulating levels of visfatin, or local concentrations of visfatin in either omental fat or placenta. Samples of female omental fat, blood and placenta were collected over a 2-year period and frozen at −80 °C until they were employed in a series of various assays. Samples were collected during delivery in pregnant women, at hysterectomy in lean women and at bariatric surgery in obese and obese diabetic women. Visfatin expression and concentrations were measured in four cohorts of women: lean controls, pregnant women at term, obese (BMI > 40) and obese diabetic women (BMI > 40). Visfatin expression was seven times higher in omental fat of pregnant women than in lean women. Immunohistochemistry (IHC) demonstrated that the visfatin gene transcript was translated to protein. An immunoblot confirmed that visfatin protein was much higher in pregnant women than in obese women. Serum visfatin was 20.8 ng/ml (n  = 7) in lean women as compared to 40.3 ng/ml in pregnant women (n  = 4); thus the increased visfatin mRNA levels in omental fat were not reflected in increased serum visfatin. We measured visfatin mRNA content of human placenta and found that placenta expresses substantial amounts of visfatin. GAP-DH, a housekeeping gene that is highly expressed in most human cells had a threshold value (Ct) of 20.9 versus a Ct of 22.4 for visfatin. Again, IHC confirmed that placental visfatin mRNA was translated into visfatin protein. [3H] 2-deoxyglucose transport was measured in partially differentiated 3T3-L1 preadipocytes. At a concentration of 2 nM, visfatin and insulin produced nearly identical increases in glucose transport. Taken together, these data suggest there is a selective increase in visfatin gene expression in pregnant women at term. Since visfatin also potently and efficaciously induced glucose transport in a cell culture model, any hypothetical role for visfatin in pregnancy should include the possibility that it may function in regulation of maternal/fetal glucose metabolism or distribution.
Keywords: Insulin resistance; Diabetes; Glucose transport; Omental fat; Placenta; Human; Women;

Pancreatic polypeptide is secreted from and controls differentiation through its specific receptors in osteoblastic MC3T3-E1 cells by Hiroaki Hosaka; Azusa Nagata; Tomohiko Yoshida; Takahisa Shibata; Toshitaka Nagao; Tomoaki Tanaka; Yasushi Saito; Ichiro Tatsuno (1390-1395).
Although the neuropeptide Y (NPY) family has been demonstrated to control bone metabolism, the role of pancreatic polypeptide (PP), which has structural homology with NPY and peptide YY (PYY) to share the NPY family receptors, in peripheral bone tissues has remained unknown. In the present study, we studied the regulatory roles of PP and its Y receptors using MC3T3-E1 cells, a murine transformed osteoblastic cell line, as a model for osteoblastic differentiation. We found that (1) PP mRNA was detected and increased during cell-contact-induced differentiation in MC3T3-E1 cells; (2) the immunoreactivity of PP was detected by radioimmunoassay and increased in culture medium during differentiation; (3) all the types of NPY family receptor mRNAs (Y1, Y2, Y4, Y5, and y6) were found to increase during differentiation; (4) PP stimulated differentiation in MC3T3-E1 cells in terms of ALP mRNA and BMP-2 mRNA. These findings suggested that MC3T3-E1 cells produce and secrete PP, which may in turn stimulate the differentiation of MC3T3-E1 through its specific receptors in an autocrine manner.
Keywords: Pancreatic polypeptide; Y receptor; MC3T3-E1 cells; Differentiation;

Estradiol and neuropeptide Y (intra-lateral septal) reduce anxiety-like behavior in two animal models of anxiety by Jorge I. Olivera-Lopez; Miguel Molina-Hernández; N. Patricia Tellez-Alcántara; M. Teresa Jaramillo (1396-1403).
Anxiolytic-like effects of intra-lateral septal nuclei (LSN) infusions of the neuropeptide Y (NPY) alone or combined with estradiol benzoate were assessed in ovariectomized Wistar rats in two animal models of anxiety-like behavior. In a conflict test, immediately punished responses were assessed: 17-β-estradiol (50.0 μg/rat, P  < 0.05) plus vehicle (intra-LSN) or intra-LSN infusions of NPY (2.5 μg/μl, P  < 0.05; 3.0 μg/μl, P  < 0.05) plus vehicle (systemic route) or the combination of subthreshold doses of 17-β-estradiol (25.0 μg/kg) plus intra-LSN infusions of NPY (2.0 μg/μl, P  < 0.05) increased the amount of immediately punished reinforcers. In the elevated plus-maze test several spatial-temporal variables were evaluated: 17-β-estradiol (50.0 μg/kg, P  < 0.05) plus vehicle (intra-LSN) or intra-LSN infusions of NPY (2.5 μg/μl, P  < 0.05; 3.0 μg/μl, P  < 0.05) plus vehicle (systemic route) or the combination of subthreshold doses of 17-β-estradiol (25.0 μg/kg) plus intra-LSN infusions of NPY (2.0 μg/μl, P  < 0.05) produced anxiolytic-like actions without affecting locomotion. It is concluded that estradiol or NPY may produce anxiolytic-like actions and that subthreshold doses of estradiol and subthreshold doses of NPY when combined produced anxiolytic-like actions.
Keywords: Conflict behavior; Elevated plus-maze; Estradiol; Lateral septal nuclei; Neuropeptide Y;

GABAA signalling is involved in N/OFQ anxiolytic-like effects but not in nocistatin anxiogenic-like action as evaluated in the mouse elevated plus maze by Elaine C. Gavioli; Filipe S. Duarte; Remo Guerrini; Girolamo Calo; Giles A. Rae; Thereza C. M. De Lima (1404-1412).
Nociceptin/orphanin FQ (N/OFQ) and nocistatin are two neuropeptides originated from the same precursor prepronociceptin/orphanin FQ (ppN/OFQ). N/OFQ is the endogenous ligand of the NOP receptor, while the target of action of nocistatin is still unknown. N/OFQ modulates various biological functions, including anxiety. Conversely, nocistatin either behaves as a functional N/OFQ antagonist or evokes per se effects opposite to those of N/OFQ. Here we investigated the interaction between the anxiolytic-like effects of N/OFQ and the anxiogenic-like action of nocistatin with those evoked by GABAA receptor ligands in the mouse elevated plus maze. The anxiogenic-like effects of the GABAA receptor antagonist pentylenetetrazol (20 mg/kg; intraperitoneal, i.p.) were abolished by the co-treatment with N/OFQ (10 pmol; intracerebroventricular, i.c.v.) while potentiated by the administration of nocistatin (0.01 pmol; i.c.v.). The anxiolytic-like effects of the benzodiazepine receptor agonist diazepam (0.75 mg/kg, i.p.) were reversed by nocistatin (0.1 pmol; i.c.v.), whereas signs of sedation were observed when mice were co-treated with diazepam and N/OFQ (3 pmol). Interesting enough, the i.p. treatment with flumazenil (1 mg/kg) blocked the anxiolytic-like effects of N/OFQ (10 pmol; i.c.v.), but not the anxiogenic effect elicited by nocistatin. Collectively, our findings suggest that the effects on anxiety elicited by pentylenetetrazol and diazepam can be counteracted or potentiated in the presence of N/OFQ and nocistatin. In addition, the effects on anxiety of N/OFQ, but not nocistatin, appear to be dependent on the benzodiazepine site of the GABAA receptor.
Keywords: Anxiety; Nociceptin/orphanin FQ; Nocistatin; NOP receptor; GABAA receptor ligands; Mouse;

Structure–activity relationships of bifunctional cyclic disulfide peptides based on overlapping pharmacophores at opioid and cholecystokinin receptors by Richard S. Agnes; Jinfa Ying; Katalin E. Kövér; Yeon Sun Lee; Peg Davis; Shou-wu Ma; Hamid Badghisi; Frank Porreca; Josephine Lai; Victor J. Hruby (1413-1423).
Prolonged opioid exposure increases the expression of cholecystokinin (CCK) and its receptors in the central nervous system (CNS), where CCK may attenuate the antinociceptive effects of opioids. The complex interactions between opioid and CCK may play a role in the development of opioid tolerance. We designed and synthesized cyclic disulfide peptides and determined their agonist properties at opioid receptors and antagonist properties at CCK receptors. Compound 1 (Tyr-c[d-Cys-Gly-Trp-Cys]-Asp-Phe-NH2) showed potent binding and agonist activities at δ and μ opioid receptors but weak binding to CCK receptors. The NMR structure of the lead compound displayed similar conformational features of opioid and CCK ligands.
Keywords: Multivalent ligands; Bifunctional peptides; Overlapping pharmacophores; G-Protein coupled receptors; Pain; Tolerance; NMR conformation;

Evidence for a μ–δ opioid receptor complex in CHO cells co-expressing μ and δ opioid peptide receptors by John M. Rutherford; Jiabei Wang; Heng Xu; Christina M. Dersch; John S. Partilla; Kenner C. Rice; Richard B. Rothman (1424-1431).
Based on non-competitive binding interactions we suggested that μ and δ receptors associate as a μ/δ receptor complex in rat brain. We hypothesized that the same non-competitive binding interactions observed in rat brain will be seen in CHO cells that co-express μ and δ receptors, but not in cells that express just μ or δ receptors. We used CHO cells expressing the cloned human μ receptor, cloned human δ receptor, or cloned mouse δ/human μ (“dimer cell”). Cell membranes were prepared from intact cells pretreated with 100 nM SUPERFIT. [3H][d-Ala2,d-Leu5]enkephalin binding assays followed published procedures. SUPERFIT, a delta-selective irreversible ligand, decreased [3H][d-Ala2,d-Leu5]enkephalin binding to δ receptors by ∼75% and to μ receptors by ∼50% in dimer cells. SUPERFIT treatment did not decrease [3H][d-Ala2,d-Leu5]enkephalin binding to μ cells. The IC50 values observed in SUPERFIT-treated dimer cells were: [d-Pen2,d-Pen5]enkephalin (1820 nM) and morphine (171 nM). Saturation binding experiments with SUPERFIT-treated dimer cells showed that [d-Pen2,d-Pen5]enkephalin (5000 nM) was a competitive inhibitor. In contrast, morphine (1000 nM) lowered the B max from 1944 fmol/mg to 1276 fmol/mg protein (35% decrease). Both [d-Pen2,d-Pen5]enkephalin and morphine competitively inhibited [3H][d-Ala2,d-Leu5]enkephalin binding to SUPERFIT-treated μ cells. The results indicate that the μ–δ opioid receptor complex defined on the basis of non-competitive binding interactions in rat brain over 20 years ago likely occurs as a consequence of the formation of μ–δ heterodimers. SUPERFIT-treated dimer cells may provide a useful model to study the properties of μ–δ heterodimers.
Keywords: Opioid receptor; Dimerization; GPCR; Morphine; Ligand binding;

Neuronal interaction between melanin-concentrating hormone- and α-melanocyte-stimulating hormone-containing neurons in the goldfish hypothalamus by Sei-Ichi Shimakura; Kenji Kojima; Tomoya Nakamachi; Haruaki Kageyama; Minoru Uchiyama; Seiji Shioda; Akiyoshi Takahashi; Kouhei Matsuda (1432-1440).
Intracerebroventricular (ICV) administration of melanin-concentrating hormone (MCH) inhibits food intake in goldfish, unlike in rodents, suggesting that its anorexigenic action is mediated by α-melanocyte-stimulating hormone (α-MSH) but not corticotropin-releasing hormone. This led us to investigate whether MCH-containing neurons in the goldfish brain have direct inputs to α-MSH-containing neurons, using a confocal laser scanning microscope, and to examine whether the anorexigenic action of MCH is also mediated by other anorexigenic neuropeptides, such as cholecystokinin (CCK) and pituitary adenylate cyclase-activating polypeptide (PACAP), using their receptor antagonists. MCH- and α-MSH-like immunoreactivities were distributed throughout the brain, especially in the diencephalon. MCH-containing nerve fibers or endings lay in close apposition to α-MSH-containing neurons in the hypothalamus in the posterior part of the nucleus lateralis tuberis (NLTp). The inhibitory effect of ICV-injected MCH on food intake was not affected by treatment with a CCK A/CCK B receptor antagonist, proglumide, or a PACAP receptor (PAC1 receptor) antagonist, PACAP(6–38). ICV administration of MCH at a dose sufficient to inhibit food consumption also did not influence expression of the mRNAs encoding CCK and PACAP. These results strongly suggest that MCH-containing neurons provide direct input to α-MSH-containing neurons in the NLTp of goldfish, and that MCH plays a crucial role in the regulation of feeding behavior as an anorexigenic neuropeptide via the α-MSH (melanocortin 4 receptor)-signaling pathway.
Keywords: Goldfish; MCH; α-MSH; Anorexigenic action; Neuronal interaction;

Anatomy, function and regulation of neuropeptide EI (NEI) by Jackson Bittencourt; María Ester Celis (1441-1450).
This review is focused on the anatomy, role and behavior of neuropeptide-glutamic acid-isoleucine (NEI), providing a general report on the neuropeptide. In addition to hormone release, this peptide also takes part in the regulation of grooming behavior and locomotor activity. NEI is produced by cleavage of prepro-MCH that probably takes place at the Lys129-Arg130 and Arg145-Arg146 sites (the glycine residue on the C-terminus of NEI strongly suggests that this peptide is amidated). This same prohormone is also the precursor of MCH, widely studied in relation to food and water intake, and NGE, of which little is known. NEI and MCH are extensively colocalized throughout the central nervous system (CNS), and NEI is also present in peripheral tissues. The latter is also effective in stimulating luteinizing hormone (LH) release and, to a lesser extent, FSH from primary pituitary cell cultures. In addition to releasing LH from the medial eminence, NEI also acts directly on gonadotropes. Lastly, this neuropeptide also acts at the CNS level on gonadotropin-releasing hormone (GnRH) neurons.
Keywords: Neuropeptide-glutamic acid-isoleucine (NEI); Anatomy; Physiology; Behavior; Hormones; Regulation: CNS; Immunoreactivity; Rats; Neurotransmitters;

Activation of endothelial nitric oxide synthase is critical for erythropoietin-induced mobilization of progenitor cells by Anantha Vijay R. Santhanam; Livius V. d’Uscio; Timothy E. Peterson; Zvonimir S. Katusic (1451-1455).
The present study aimed to define the ability of erythropoietin (EPO) to mobilize hematopoietic stem cells (c-kit+/sca-1+/lin-1; KSL-cells) and hematopoietic progenitor cells (CD34+ cells), including vascular endothelial growth factor receptor 2 expressing hematopoietic progenitor cells (CD34+/Flk-1+ cells). We also sought to determine the role of endothelial nitric oxide synthase (eNOS) in EPO-induced mobilization. Wild type (WT) and eNOS−/− mice were injected bi-weekly with recombinant erythropoietin (EPO, 1000 U/kg, s.c.) for 14 days. EPO increased the number of KSL, CD34+, CD34+/Flk-1+ cells in circulating blood of wild type mice. These effects of EPO were abolished in eNOS−/− mice. Our results demonstrate that, EPO stimulates mobilization of hematopoietic stem and progenitor cells. This effect of EPO is critically dependent on activation of eNOS.

Novel brevinins from Chinese piebald odorous frog (Huia schmackeri) skin deduced from cloned biosynthetic precursors by Zhenzhen Quan; Mei Zhou; Wei Chen; Tianbao Chen; Brian Walker; Chris Shaw (1456-1460).
Antimicrobial peptides represent the most characterized and diverse class of peptides within the defensive skin secretions of anuran amphibians. With an ever expanding database of primary structures, the current accepted rules for nomenclature have become increasingly difficult to apply to peptides whose primary structural attributes are either unique or that fall between those that define existing groups. An additional factor that adds to the confusion is the regular re-classification or revision of existing taxa. In the present study, we have identified five new antimicrobial peptide homologs in the defensive skin secretion of the Chinese piebald odorous frog, Huia schmackeri (formerly Rana (Odorrana) schmackeri), by cloning of their respective biosynthetic precursors. As these peptides are obvious homologs of the brevinin-1 and brevinin-2 families we have named these in accordance: (1) brevinin-1HS1, (2) brevinin-2HS1, (3) brevinin-2HS2, (4) brevinin-2HS3 and (5) brevinin-1HS2. The reasons for adopting these names are discussed. It is clear that with an ever-increasing number of amphibian skin antimicrobial peptides appearing in the literature that a consistent nomenclature scheme needs to be established.